1. Field of the Invention
The present invention relates to a method and apparatus for assay in utilizing attenuated total reflection, and a sample immobilizing device. More particularly, the present invention relates to a method and apparatus for assay in utilizing attenuated total reflection, and a sample immobilizing device, in which a sample can be assayed reliably by keeping fluidity in a flow channel according to a surface plasmon resonance (SPR) biosensor system, and also a sample immobilizing device.
2. Description Related to the Prior Art
An assay apparatus in utilizing attenuated total reflection for assaying a sample is known in the field of the biosensor. A thin film, or metal film, is formed on a transparent dielectric medium. One surface of the metal film is a sensing surface where reaction of a sample is occurs. Another surface of the metal film is a light entrance surface where light is applied by satisfying a condition of total reflection. The reaction is detected to assay the sample according to attenuation of the reflected light from the light entrance surface. U.S. Pat. No. 5,313,264 (corresponding to JP-A 4-501462) discloses a surface plasmon resonance (SPR) sensor as a typical example for this assay.
In a metal, free electrons vibrate to generate the compressional wave called a plasma wave. Surface plasmon is a term to mean the compressional wave created on the surface of the metal and included in plasmon as quantized expression of the compressional wave. The surface plasmon travels along the surface of the metal. The surface plasmon resonance (SPR) assay apparatus is constructed to detect surface plasmon resonance- created on the sensing surface which is a first surface of the metal film.
Light for detection is applied to a light entrance surface of the metal film that is back to the sensing surface so that the total reflection condition is satisfied, namely at an angle of incidence equal to or more than a critical angle. In addition to the total reflection created on the light entrance surface, a small component of the light passes through the metal film without reflection, and penetrates to the sensing surface. A wave of the penetrating component is called an evanescent wave. Surface plasmon resonance (SPR) is created as when frequency of the evanescent wave coincides with that of the surface plasmon. In response to this, intensity of the reflected light attenuates remarkably. In the assay apparatus, the attenuation in the reflected light reflected by the light entrance surface is detected, to recognize creation of the SPR on the sensing surface.
The angle of incidence, namely resonance angle of the light to generate the SPR depends on the refraction index of the transmission medium transmitting evanescent wave and surface plasmon. In other words, a change in the resonance angle to create SPR changes in response to a change in the refraction index of the transmission medium. The substance contacting the sensing surface is a transmission medium transmitting the evanescent wave and surface plasmon. If binding or separation between two molecules occurs on the sensing surface, the resonance angle changes because of a change in the refraction index of the transmission medium. In the SPR system, the change in the refraction index is detected, to measure interaction of molecules.
The assay apparatus can be used for various kinds of studies in a biochemical field or the like, for example to study interaction of protein, DNA and various biochemical substances, and to select candidate drugs by screening. It is possible to use one of two substances as a ligand and another one of them as an analyte if those have bioaffinity. For the purpose of screening, protein as biomaterial is used as ligand. Candidate drugs are discretely used as analyte, and contacted with the ligand on the sensing surface, to study interaction.
JP-A 6-167443 and U.S. Pat. No. 5,822,073 disclose an SPR assay apparatus in which an optical system of Kretschmann configuration is used for incidence of light to the metal film. According to the Kretschmann configuration, the light entrance surface of the metal film is fitted on a prism, which condenses light and directs the light to the light entrance surface in a manner conditioned for total reflection. A sample or ligand is immobilized on the sensing surface. A flow channel is formed to have the sensing surface inside, and causes analyte liquid to flow. The analyte liquid is introduced in the flow channel to flow, and is caused to contact the ligand. Interaction between the analyte liquid and the ligand is assayed by detecting surface plasmon resonance created during the reaction.
JP-A 6-167443 discloses an assay stage disposed in the apparatus casing and having a prism and a flow channel. A sensor of a chip type according to the SPR system is placed on the assay stage, the sensor including a glass base board which is dielectric and transparent, and metal film overlaid thereon. The sensor of the chip type is secured to the apparatus casing removably, and positioned so as to set the sensor surface inside the flow channel of the casing, and set the light entrance surface on the prism. Prior to the assay, it is necessary in a pre-treatment to immobilize ligand on metal film of the sensor of the chip type. This is ligand immobilization. According to the SPR system of JP-A 6-167443, the sensor of the chip type is kept mounted on the assay stage.
At first before the assay, the sensing surface of the sensor of the chip type appears externally. A portion of the flow channel at the sensing surface is open. When the sensor of the chip type is mounted on the assay stage, the sensing surface covers and encloses the open portion of the flow channel. This enables introduction of liquid to the flow channel. Ligand liquid is introduced to the flow channel, and ligand is immobilized. After this, analyte liquid is introduced before assay is made.
However, there is a problem in the above assay by use of the sensor of the chip type. The assay process directly follows the immobilization for one sensor. Efficiency or throughput in the operation of the assay system cannot be raised if the immobilization is made for a plurality of the sensor of the chip type. The same assay stage is used for either of the immobilization and the assay process. The assay process would not be conducted during the immobilization for next sample. The immobilization has a low speed of a rate-determining level defining the low speed of the entire assay system even though the assay process is possible in a quick manner. Suggestions have been made for improving efficiency in the immobilization which requires much more time than the assay process.
It is conceivable to raise throughput of the assay by the immobilization for plural sensors together, and by serially assaying plural sensors after the immobilization. The sensor are mounted on the assay stage and subjected to the immobilization. Each one of the sensors after the immobilization is removed from the assay stage. Other sensors are then mounted on the assay stage for the immobilization. By repeating such a sequence, the immobilization is collected for the plural sensors. After this, the sensors after the immobilization are mounted on the assay stage one after another, and subjected to the assay. Thus, the immobilization can be completed together before the assay in the collective manner irrespective of time required by the immobilization. Improvement in the throughput of the assay might be expected.
If the sensor of the chip type is removed from the assay stage, the sensing surface dries by volatilization because uncovered externally. The sensing surface cannot be kept wet. Molecules or other substances as ligand, for example protein, are likely to degrade by drying in relation to initial characteristic, for example properties as enzymes and affinity for binding with other substances. It is impossible to recognize the properties accurately if drying proceeds after the immobilization until the assay.
Even if a substance does not change functionally upon drying, the substance comes in a state not returning to an initial state even when liquid is abruptly provided. No reaction will occur upon the start of the onset. Namely, a signal output is unstable. This lengthens the time of assay, because sufficient time is required for stability in the output signal.
No known technique solves those problems in the assay due to temporary drying of ligand after the immobilization until the onset of assay.